The primary goal of our research is to understand the mechanisms involved in DNA replication in eukaryotic cells. We study these processes using DNA-containing viruses as models, since this affords the opportunity to not only gain new insights into the process of DNA replication itself, but also to learn more about the biology of important pathogens. In our work on herpes simplex virus we have focused on two virus-encoded proteins that are known to be essential for the replication of the viral genome: the helicase primase complex, comprised of three subunits (UL5, UL8, and UL52), and the origin binding protein (UL9). In our studies on the helicase-primase complex, we have used the yeast two-hybrid system to map out the regions of the three polypeptides that are important for interaction among the three subunits. These studies have located domains of each of the three polypeptides that are critical for interaction with the other two subunits, and we are currently analyzing a number of point mutant derivatives of each protein to locate the critical amino acid residues. We have also used the yeast two-hybrid system to attempt to identify cellular proteins that interact with the origin binding protein, UL9.. Initial screening of a cDNA library for proteins that interact with UL9 has been completed and a number of cDNAs encoding interacting proteins have been identified. Sequencing of these cDNAs for possible identification is in progress. In our work on vaccinia virus DNA replication, we are attempting to develop an assay by which the genetics of vaccinia DNA replication can be systematically analyzed. The assay involves transfecting cells with all of the viral genes that might be involved in DNA replication based on the current understanding of the function of all vaccinia genes. Our current estimate is that there are about 20 genes that encode proteins that are known to be involved in DNA replication (such as DNA polymerase) or whose function is completely unknown and therefore cannot be ruled out. These genes are being cloned into plasmid vectors containing the promoter for phage T7.